The article explores methods to boost biohydrogen production from cheese whey through the lactate metabolic pathway using dark fermentation. Initial testing in batch configuration led to the determination of maximum hydrogen yield and consumption efficiency. The study then transitioned to continuous operation with various phases, emphasizing the importance of the support medium in stabilizing the system. Addition of Fe2O3 nanoparticles at specific concentrations notably increased hydrogen productivity. Analysis showed a positive correlation between butyrate concentrations and enhanced hydrogen production rates. Furthermore, the presence of iron-based nanoparticles effectively regulated lactate concentration, maintaining it at optimal levels. The investigation into bacterial community dynamics highlighted a mutually beneficial interaction between lactic acid bacteria and hydrogen-producing bacteria. Prevotella, Clostridium, and Lactobacillus were identified as predominant genera. The study underscores the potential of nanoparticle addition in enhancing biohydrogen productivity via lactate-driven dark fermentation, offering insights into sustainable bioenergy production from organic waste streams.